Ecotoxicological evaluations of common hatchery substances and procedures used in the production of Sydney rock oysters Saccostrea glomerata (Gould 1850).

ABSTRACT Progress in the Sydney rock oyster Saccostrea glomerata industry, through the adoption of oyster spat selected for faster growth and disease resistance, has been hampered by long-term variability in commercial hatchery spat supply. As part of a broader study to evaluate spat production impediments, the chronic toxicity of substances commonly used in bivalve hatcheries and the effects of handling procedures during early ontogeny (embryo to D-veliger) were evaluated. Among the substances tested, chlorine, Virkon S and Virkon S for Aquaculture (virucidal disinfectants, Antec International Limited, Suffolk, UK), bore water and stored rainwater were found to significantly affect larval development at practically/commercially-relevant concentrations. Toxicity was determined by quantifying embryo-larval development after 48 h exposure and three tests were performed for each substance or procedure. Concentrations of 0.83-1.66 mg [L.sup.-1] of chlorine in seawater and 0.05-0.5 mg [L.sup.-1] of Virkon S in seawater significantly decreased the normal development of embryos after 48 h exposure. An [EC.sub.50] value of 0.76-1.18 mg [L.sup.-1] for chlorine in seawater and 0.47-1.01 mg [L.sup.-1] for Virkon S in seawater was derived. The [EC.sub.50] value for Virkon S for Aquaculture was 0.99-1.12 mg [L.sup.-1] and this substance caused significant development problems for larvae at a concentration of 0.5 mg [L.sup.-1] in seawater. Tests that added stored rainwater to seawater had a significant decrease in the percentage of embryos developing to the D-veliger stage at concentrations greater than 1%, whilst no effect was detected at 0.1%. The [EC.sub.50] value for rainwater was 0.67% to 2.29%. Similarly, bore water added to seawater caused a significant decrease in the percentage of embryos to develop to the D-veliger stage at a concentration of 10% and no effect was observed at 1%. The [EC.sub.50] value for bore water ranged between 2.3 and 3.7%. Handling procedures for screening fertilized eggs did not significantly decrease development percentage after 48 h incubation time. Likewise, tests conducted with de-ionized water at concentrations up to 10% added to seawater did not significantly reduce the percentage of embryos to develop to the D-veliger stage after 48 h exposure. This study highlights the sensitivity of S. glomerata larvae to surfactants and disinfectants and identified contaminated water sources.

KEY WORDS: rock oyster, toxicity, Saccostrea glomerata, embryo-larval development, Virkon S, chlorine

INTRODUCTION

Production of the Sydney rock oyster, Saccostrea glomerata (Gould 1850) occurs in estuaries predominately along the Australian east coast (Nell 1993) and is one of the oldest and largest aquaculture industries in the eastern states. In New South Wales (NSW), the industry currently generates approximately US$27 million in sales annually (NSW Department of Primary Industries 2004), although overall production has decreased since the mid 1970s. The causes of the decline are varied and include competition from other faster growing oyster species, disease and declining water quality (White 2002).

To assist industry, NSW Department of Primary Industries (DPI) commenced breeding programs for S. glomerata in 1990 with the intention of producing faster growing oysters and later incorporating disease resistance. This ongoing program has developed lines of S. glomerata capable of reaching market size (50 g) 12.5 mo faster than control oysters (Nell & Perkins 2005) and lines that are resistant to QX disease (Nell & Perkins 2006). Industry adoption of the benefits of genetic research have however been greatly constrained by the chronic failure of attempts to produce commercial quantities of S. glomerata in hatcheries in NSW.

Hatchery production of S. glomerata commenced in the early 1980s but has been plagued by recurrent mass mortality syndromes in both larvae and spat. In a review of hatchery production attempts for S. glomerata between 1985 and 1990, Heasman et al. (2000) found 57% of larval runs failed within eight days of fertilization. To overcome these failures, a systematic program has begun to identify problems, among which toxic agents and stressful handling procedures were identified as a potential source of larval mortality.

Oyster embryos are sensitive and vulnerable to a range of pollutants, particularly trace metals (Wisely & Blick 1967, Calabrese et al. 1973, Calabrese et al. 1977, Martin et al. 1981, Wilson & Hyne 1997). Also, seawater and freshwater supplies for hatcheries can become contaminated in a number of ways by many factors related to materials, design or location as well as the origin of the water or the presence of deleterious microflora. Moreover, toxins that cause hatchery problems can be difficult to identify and temporal (Jones 2003).

Cleaning and disinfection of water, tanks, equipment, and facilities used for rearing bivalves is performed by filtration, desiccation, and chemical application processes; and it is common practice in shellfish hatcheries to maintain hygiene. Problems can arise when small amounts of chemicals unintentionally find their way into rearing tanks, particularly tanks used for early larval culture. Handling procedures also have the potential to impact embryos either through physical damage, air exposure or dramatic temperature and salinity fluctuations. For example, physical damage may occur when eggs and sperm are screened through fine PVC mesh (20-80 [micro]m) to remove debris and feces collected from the spawning table.

This study evaluated the chronic toxicity of substances commonly used in oyster hatcheries and the effect of egg screening on embryonic development of S. glomerata. Substances to which embryos or larvae could be inadvertently or accidentally exposed were tested for toxicity in preliminary trials. These substances and their usage are listed in Table 1. Particular substances found to be toxic were then classified as "high" risk and subjected to more rigorous ecotoxicological testing. The impacts of Virkon S and Virkon S for Aquaculture (virucidal disinfectants, Antec International Limited. Suffolk, UK) as well as chlorine to S. glomerata embryos were quantified. Other contaminants that could impact on larval development were investigated and included: rainwater: bore water, and algal culture media used in bivalve hatcheries.

MATERIALS AND METHODS

Spawning

Rock oyster embryos for the preliminary range finding tests were obtained from broodstock collected from the Manning River (31[degrees]53'S, 152[degrees]41'E) and spawned on 2 June 2004. Embryos for the definitive tests were obtained from two spawning inductions, the first on August 17, 2004 and the second on August 24, 2004. Broodstock for the definitive tests were from Port Stephens (32[degrees]11'S, 152[degrees]20'E) and Wagonga Inlet (36[degrees]13'S. 150[degrees]8'E) and were collected on the 28 June 2004 and 27 July 2004, respectively.

Thirty adult broodstock oysters were used for each spawning. Oysters were scrubbed to remove mud, cleaned of fouling organisms and held at the Port Stephens Fisheries Center (PSFC) bivalve hatchery in a conditioning unit at 25[degrees]C and 34 ppt before each test. The S. glomerata embryos for tests were obtained using thermal stimulation and salinity fluctuations in natural seawater (22[degrees]C, 33 ppt and filtered to 1 [micro]m) following the spawning procedure described in Frankish et al. (1991). This procedure was used in favor of physically stripping gametes from the gonads of oysters as it reduces the possibility of using under-developed gametes in experimental treatments (His et al. 1997).

Oysters releasing gametes were placed into separate 500 ml plastic containers partially filled with seawater (22[degrees]C, 33 ppt and filtered to 1 [micro]m). Eggs from at least six females were pooled, passed through a 55 [micro]m screen to remove debris and then placed into a 1 L plastic beaker for each test. The number of eggs was estimated using a Sedgewick-Rafter counting chamber and fertilized with simultaneously released sperm from at least three males for each test. The egg and sperm suspension was left for another 20 min, while fertilization occurred, and then stocked into the test vessels.

Embryo Development Tests

A static 48 h larval abnormality test was used. Tests were conducted according to methods and procedures described in ASTM (1994) and Krassoi et al. (1996). A series of range finding tests were conducted initially using the substances and procedures listed in Table 1. The information from these tests was then used to design and carry out three definitive tests, which had five replicates per treatment. The test control and diluent water was natural seawater that had a salinity level of 34 [+ or -] 0.5 ppt and was filtered to 1 [micro]m. Daylight and a fluorescent lighting (<1000 lux) were used to maintain a photoperiod of 16 h of light and 8 h of darkness over the experimental period. Test vessels were 120 mL polypropylene vials that contained 100 mL of test solution after the toxicant and larvae were added. Temperature, pH, dissolved oxygen concentration and salinity of additional treatments were measured at the start and conclusion of the experiment. Fertilized eggs were added at a density of 20 eggs [mL.sup.-1] to each test chamber using an automatic pipette. This egg density was lower than the egg density used by Krassoi (1995) (100 eggs [mL.sup.-1]) and is not known to cause abnormality. Test chambers were randomly placed in a temperature controlled incubator chamber at 25 [+ or -] 0.5[degrees]C immediately after fertilized eggs were added. Fertilization rate was verified using light microscopy (x 250 magnification) on an additional test chamber removed from the experiment after 3 h.

The test parameter was the proportion of abnormalities at the D-veliger stage. Most S. glomerata embryos have reached D-veliger stage after 20 h incubation time at 34 ppt salinity and 25[degrees]C. Tests were however continued for 48 h before 0.8 mL of 10% buffered formalin was added to each test unit to halt development. Normal D-veliger larvae are characterized by a straight hinge and symmetrical shell plates. Larvae were considered abnormal if they were deformed, had misshapen or asymmetrical shell plates or had not progressed past the trochophore or blastophore stage (undeveloped) after 48 h had elapsed (Krassoi 1995, Krassoi et al. 1996, His et al. 1997). Typical examples of normal straight-hinge larvae, abnormal larvae and an undeveloped larva are displayed in Figure 1. A total of 100 larvae (5%) from each test unit were randomly selected and examined for the percentage of D-veliger development using light microscopy (x 100 magnification).

[FIGURE 1 OMITTED]

Toxicants

The preliminary range finding tests evaluated the substances and egg screening procedure listed in Table 1. The exposure levels used in these tests for each substance or procedure are recorded in Table 2. The substances classified as "high" risk (Table 1) and found to have a toxic effect at plausible concentrations for contamination of rearing tanks were then selected for the definitive test and included: rainwater; bore water; chlorine; Virkon S; and, algal supernatant. Concentrations were adjusted for the definitive test based on results from the preliminary test so that no abnormal development occurred in the lowest concentration and 100% abnormal development occurred at the highest concentration. The toxicity of Virkon S for Aquaculture, a virucidal disinfectant for aquacultural applications, to S. glomerata was evaluated at a later date (May 1, 2006) as this product was not in use when the definitive tests were done. The concentrations of toxicants tested in the definitive test are listed in Table 3. De-ionized water was used to make stock solutions of Virkon S and Virkon S for Aquaculture to add to the test vessels. Algal supernatant was prepared by combining Tahitian Isochrysis aff. galbana, Pavlova lutheri, and Chaetoceros calcitrans in the proportions that are fed to S. glomerata D-veliger larvae (1:1:2, dry weight) and placing the mixture in a centrifuge before decanting the supernatant. Additionally, de-ionized water was included in the definitive test at the same concentrations as bore water and rainwater to examine osmotic effects from decreased salinity levels on embryo development. Handling procedures were also of concern and the effect of repeated screening of fertilized eggs was tested. Fertilized eggs were passed through a 55 gm mesh screen 4, 8, and 16 times before stocking into the test chambers.

Data Analyses

The proportion of abnormal larvae was determined for all tests conducted. The mean % abnormal development data were calculated by combining data from the five replicate treatments within each of the three definitive tests and these data were corrected for the pooled percentage abnormality in the control treatments. The [EC.sub.50] value and the 95% confidence intervals were calculated using the trimmed Spearman Karber method (Hamilton et al. 1977) on corrected abnormality data for the tests involving rainwater, chlorine, Virkon S, Virkon S for Aquaculture, bore water, and algal supernatant. Proportion larval abnormality data were arcsine transformed and analyzed using Analysis of Variance (ANOVA) and Dunnett test to determine the no observable effect concentration (NOEC) and lowest observable effect concentration (LOEC) following Bartlett test for homogeneity of variance. The Dunnett Program Version 1.5 computer program obtained from Environmental Monitoring Systems Laboratory, United States Environmental Protection Agency was used to perform these analyses.

RESULTS

The water quality variables of pH, electrical conductivity, dissolved oxygen concentration, and temperature remained static in the test chambers during the test period. However, adding Virkon S, Virkon S for Aquaculture, de-ionized water, rainwater, and bore water altered diluent water quality. The addition of Virkon S and Virkon S for Aquaculture caused the pH of the test chambers to decrease by 0.4 units when added at a concentration of 50 mg [L.sup.-1]. De-ionized water, rainwater, and bore water caused a decrease of 4.3 dS [m.sup.-1] in the electrical conductivity over the concentrations tested (0-10%) but did not alter the pH compared with the control treatment.

The proportion of abnormal larval development determined from preliminary testing is listed in Table 2. These results indicate that chlorine, potassium permanganate, Virkon S, Antec Cleaning, and Sanitizing Agent (DSC-1000), Pyroneg, sodium thiosulfate, rainwater, bore water, algal supernatant, and egg screening have the potential to impact S. glomerata development.

The acute toxicities of substances in the definitive test that impact on S. glomerata embryonic development are listed in Table 3. Embryonic development of S. glomerata when exposed to rainwater, bore water, de-ionized water, algal supernatant, chlorine, Virkon S, Virkon S for Aquaculture or repeated screening are displayed in Figure 2. Rainwater was investigated over the range of 0.01% -10% in seawater. The LOEC for rainwater used in this study was 1% and the [EC.sub.50] value was estimated to be between 0.67% and 2.29%. Bore water was investigated over the same concentration range as rainwater and had a lesser impact on embryonic development compared with rainwater. The LOEC and [EC.sub.50] range for bore water was 10% and 2.30%-3.70%, respectively. Heterogeneity of variances was detected using Bartlett's test in Test 3 for rainwater and bore water. Tests 1 and 2 had homogenous variances and provided the same LOEC and NOEC levels as Test 3 for both toxicants.

To test for osmotic effects from decreased salinity levels on embryo development, de-ionized water was tested at concentrations of 0.01, 0.1, 1, and 10% in seawater. ANOVA did not detect a significant difference in embryonic development across the four concentrations in the three tests (F = 0.428; df = 4/10; P > 0.05). Likewise, screening of fertilized eggs was found to not have a significant effect on embryonic development (F = 1.651; df = 3/8; P > 0.05). Figure 2 shows the proportion of abnormally developed larvae resulting from the de-ionized water and egg screening treatments.

The [EC.sub.50] value estimated for algal supernatant ranged between 6.51 and 17.80%. The LOEC and NOEC levels for algal supernatant were different in the third test from the first two tests conducted. The LOEC level was 20% in Tests 1 and 2 and 10% in Test 3. The NOEC in test 3 was 1%, however it was estimated to be 10% in the first two tests. The NOEC result from the third test was 1% and is much greater than the concentration of algal mixture typically added to an incubation tank. Heterogeneity of variances was detected in the third test.

Embryonic development of Rock oysters was inhibited when exposed to treatments containing chlorine. No development to the D-veliger larval stage occurred at an initial concentration of 2.08 nag [L.sup.-1] in the three tests (Fig. 2). Chlorine levels at initial concentrations of 0.83 mg [L.sup.-1] in Test 1 and 1.66 mg [L.sup.-1] in Tests 2, and 3 were observed to have a significant (P < 0.05) effect on embryonic development. The range of the estimated [EC.sub.50] value was from 0.76-1.18 mg [L.sup.-1] for the three tests. Likewise, Virkon S caused significant (P < 0.05) levels of abnormal development in S. glomerata embryos at a concentration of 0.05 mg [L.sup.-1] in tests 3 and 5 and 0.5 mg [L.sup.-1] in tests 1, 2, and 4. The LOEC and NOEC determined for Virkon S for Aquaculture in both tests conducted was 0.5 mg [L.sup.-1] and 0.05 mg [L.sup.-1], respectively. Concentrations of Virkon S and Virkon S for Aquaculture in seawater above 50 mg [L.sup.-1] prevented all embryos developing to the D-veliger larval stage (Fig. 2). Heterogeneity of variances were detected in the data used for Tests 1 and 3 for chlorine, Tests 1 and 4 for Virkon S and Test 1 for Virkon S for Aquaculture. However, these results are comparable to tests where there was equality of variances according to Bartlett test.

DISCUSSION

A range of pollutants can cause problems to larval batches in hatcheries, including: metals; disinfectants; surfactants; hydrocarbons; endocrine disruptors; and antibiotics (Jones 2003). Generally, these substances are accidentally or inadvertently introduced to larval cultures, however given the various pathways that contaminants can enter hatcheries, great care is required.

Virkon S and chlorine solutions can both be used for cleaning and disinfection of larval rearing equipment. Virkon S is a virucidal disinfectant that contains potassium peroxomonosulphate, sulphamic acid and sodium alkyl benzene sulphohate. Virkon S is used at a concentration of 5 g [L.sup.-1] and is applied to tanks used for larval and spat rearing (Table 1) between water changes, conducted on alternate days. There is negligible information available on the toxicity of Virkon S to aquatic organisms in the literature. One investigation of the toxicity of Virkon S to post larval (15 day old) giant tiger prawns and adult giant tiger prawns determined the [LC.sub.50] for this substance to be 10.31 mg [L.sup.-1] and 120.33 mg [L.sup.-1], respectively, and all post larval prawns were dead after exposure to 50-mg [L.sup.-1] for 5 h (Suphamat 2005).

Small concentrations of Virkon S or chlorine have a significant effect on embryonic development. In the case of Virkon S, catastrophic contamination of incubation tanks can occur through careless use or failure to rinse the chemical residue away following application. Approximately 500 mL of Virkon S prepared at the recommended concentration (Table 1) is applied to clean a 1,000 L incubation tank before fertilized eggs are added and between regular water changes during larval rearing. If the tank was not properly rinsed, the concentration of Virkon S could be as high as 2.5 mg [L.sup.-1], which could prevent more than 80% of S. glomerata embryos from developing to the D-veliger stage. This level of non-development is not acceptable for research or production runs where generally more than 95% of S. glomerata embryos develop into D-veliger larvae.

Tests conducted on May 1, 2006 showed that Virkon S for Aquaculture and Virkon S impacted S. glomerata development in a similar way. The constituents of Virkon S for Aquaculture are different from Virkon S and are pentapotassium bis(peroxymonosulphate) bis(sulphate), sulphamidic acid, sodium dodecylbenzenesulfonate, and dipotassium peroxidisulphate. Both products are virucidal disinfectants and are used for the same applications in a hatchery environment (Table 1).

Applied as sodium hypochlorite (LR), chlorine is used at a concentration of between 30 and 50 mg [L.sup.-1] to clean and disinfect larval rearing equipment. Chlorine must also be carefully handled in areas where oysters are spawned and where larvae are incubated. It is also important that any equipment cleaned using chlorine, particularly screens, be thoroughly rinsed before coming into contact with eggs or embryos. Ideally, the use of solutions containing chlorine should be restricted to areas of the hatchery isolated from areas used to rear larvae to minimize the risk of accidental contamination. Furthermore, neutralization of chlorine with thiosulfate also can pose a potential hazard. The preliminary tests conducted for this study indicated that thiosulfate could restrict S. glomerata development.

[FIGURE 2 OMITTED]

Stewart et al. (1979), Roosenburg et al. (1980), and Richardson et al. (1982) investigated the effects of chlorination on Crassostrea virginica larvae. These studies were conducted to investigate pollution of the natural environment by chlorinated discharges originating from industrial and sewage sources and identified impacts to larval development as a result of exposure to chlorinated waters. In addition to this, residual oxidants from chlorinated seawater are also particularly toxic to oyster larvae (Stewart et al. 1979, Roosenburg et al. 1980) and other aquatic organisms and should always be neutralized before release from hatchery facilities (OIE 2003).

Contamination may also occur because of hatchery design, location, or materials used in construction (Jones 2003). Stored water sources used in hatcheries can be contaminated at a number of different points. Contamination can occur during collection of the water, during transport or pumping of water to a storage tank, in the tank itself and in the pipes that deliver the water to the hatchery.

The PSFC bivalve hatchery is reliant on stored rainwater or bore water for freshwater, which is used for rinsing, cleaning, and to dilute seawater, predominately for experiments conducted within the hatchery. Both rainwater and bore water are supplied to the hatchery through copper pipe plumbing. The quality of the rainwater supply to the hatchery is highly variable and dependent on antecedent rainfall, storage time, and the amount of interaction with organic matter accumulated on the roof catchment and in the storage tanks. Likewise, the quality of bore water can also be influenced by climatic factors, chemical treatment and the mineralogy of the aquifer's sediments.

The rainwater and bore water used for this study caused significant impacts to larvae at relatively low concentrations; levels as low as 1% rainwater in seawater caused a significant decrease in embryonic development. Rainwater is often used to decrease salinity levels to induce oysters to spawn. Bore water was the only feasible alternative to rainwater to reduce salinities for spawning. Bore water was found to be less toxic than rainwater to S. glomerata embryos, however, it still caused significant reductions in development at a concentration of 10% added to seawater.

The quality of rainwater and bore water is likely to vary considerably over time, however, data from this study showed that the impacts were similar on different test dates. The toxicity of the freshwater at the study location may affect experiment results, particularly those from long-term studies. Treatments that used de-ionized water in the same concentrations as bore water and rainwater did not significantly affect embryonic development. The low abnormal development rates in treatments that contained rainwater and bore water can be attributed to contaminants contained within those waters and not because of osmotic effects caused by a decreased salinity level. Rainwater had elevated levels of Cu, Mg, Na, S, and Zn; whereas bore water had elevated levels of Ca, Cu, K, Mg, Na, Si, S, and Zn compared with the de-ionized water samples (Table 4). Therefore, de-ionized water is recommended for reducing the salinity of seawater, particularly for embryo and larval rearing.

Algal mixtures typically consisting of Tahitian Isochrysis aff. galbana, Pavlova lutheri and Chaetoceros calcitrans are not commonly added to tanks containing fertilized eggs until the majority have developed to D-veliger. Regardless, these mixtures can contain low concentrations of trace metals or toxic metabolites, or if bacterized, toxins that can affect larval survival and growth (Calabrese & Davis 1970). Thus their impact on embryos was tested as an indicator of potential effects on D-veliger growth and survival.

Algal mixtures fed to larvae within the 48 h post fertilization were tested to ensure it did not inhibit development at the concentrations added for daily feeds. Approximately 2 L of algal mixture is added to incubation tanks within the initial 24 h after fertilization as a food source for rapidly developing larvae. This equates to a concentration of 0.2% algal suspension in seawater in a 1,000 L incubation tank. The data from this study showed that this concentration did not affect embryonic development.

Handling procedures of S. glomerata eggs were also of concern and the effect of screening fertilized eggs was included in this study to ensure this process did not physically damage eggs. Eggs are typically screened through a 55 [micro]m screen prior to or after sperm have been added, to remove feces and debris accumulated during spawning. Screening eggs through a 55-[micro]m mesh did not affect embryonic development.

As oysters spawn they are removed from the aquarium and placed into separate containers that hold seawater at ~34 ppt and 25[degrees]C (Frankish et al. 1991). The temperature on the spawning table varies up to 10[degrees]C during cycles and could be up to 6[degrees]C different to the container. Also, during spawning induction, the salinity level of the aquarium is decreased from 34 ppt to 20-24 ppt. Salinity and temperature shocks of this magnitude may impact S. glomerata eggs and sperm and further handling experiments are planned to investigate the effect on early larval development of temperature shocks that may occur after spawning.

The study was conducted to: maximize larval yields; increase awareness among hatchery staff of the risks posed by various substances frequently used in hatcheries; and, to establish a method for examining new substances, materials and handling procedures introduced to the hatchery. Toxicity testing conducted for this study has identified several risk factors that have the potential to negatively affect the early larval development of S. glomerata.

ACKNOWLEDGMENTS

The authors thank the staff of the Port Stephens Fisheries Research Centre for their assistance, in particular Ben Finn, Stephan O'Connor, and Lynne Foulkes for their help in the hatchery and for the provision of algae; Drs G. Allan, M. Booth, T. Gibson, and J. Sammut for valuable editorial comments during the preparation of this manuscript. This study was supported by a Fisheries Research and Development Corporation grant 2003/209.

LITERATURE CITED

A.S.T.M. 1994. E 724-94. Standard guide for conducting static acute toxicity tests starting with embryos of four species of saltwater bivalve molluscs. In: pesticides; resource recovery: hazardous substances and oil spill responses: waste disposal: biological effects, Volume 11.04, American Society for Testing and Materials, Philadelphia, pp 192-209.

Calabrese. A. & H. C. Davis. 1970. Tolerances and requirements of embryos and larvae of bivalve molluscs. Helgolander wiss. Meersunters 20:553-564.

Calabrese, A., J. R. MacInnes, D. A. Nelson & J. E. Miller. 1977. Survival and growth of bivalve larvae under heavy-metal stress. Mar. Biol. 41:179-184.

Calabrese, A., R. S. Collier, D. A. Nelson & J. R. MacInnes. 1973. The toxicity of heavy metals to embryos of the American oyster Crassostrea virginica. Mar. Biol. 18:162-166.

Frankish, K. R., L. J. Goard & W. A. O'Connor. 1991. The development of hatchery rearing techniques of the Sydney rock oyster (Saccostrea commercialis) at the Brackish Water Fish Culture Research Station, Salamander Bay, NSW, 2301. 26 pp.

Hamilton, M. A., R. C. Russo & R. V. Thurston. 1977. Trimmed Spearman-Karber method for estimating median lethal concentrations in toxicity bioassays. Environmental Science and Technology 11:714-719. Correction in. Environ. Sci. Technol. 12:417.

Heasman, M. P., L. Goard, J. Diemar & R. B. Callinan. 2000. Improved early survival of molluscs: Sydney rock oyster (Saccostrea glomerata). NSW Fisheries Final Report Series: 29. Nelson Bay. 63 pp.

His, E., M. N. L. Seaman & R. Beiras. 1997. A simplification the bivalve embryogenesis and larval development bioassay method for water quality assessment. Water Res. 31:351-355.

Jones, B. 2003. Review of hygiene, equipment and protocols. In: M. P. Heasman, editor. Proceedings, Sydney Rock Oyster Hatchery Health Workshop, August 8-9, 2002. NSW Fisheries Research Report Series: 61. Nelson Bay. pp. 45-49.

Krassoi, R. 1995. Salinity adjustment of effluents for use with marine bioassays: effects on the larvae of the doughboy scallop Chlmays asperrimus and the Sydney Rock Oyster Saccostrea commercialis. Australasian Journal of Ecotoxicology 1:143-148.

Krassoi, R., D. Everett & I. Anderson. 1996. Protocol for using doughboy scallop Chlamys asperrima (Mollusca: Pectinidae) L. to test the sublethal toxicity of single compounds and effluents. National Pulp Mills Research Program Technical Report No. 17. Canberra: CSIRO. 56 pp.

Martin. M., K. E. Osborn, P. Billig & N. Glickstein. 1981. Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae. Mar. Pollut. Bull. 12:305-308.

Nell, J. A. 1993. Farming the Sydney Rock Oyster (Saccostrea commercialis) in Australia. Reviews in Fisheries Science 1: 97-120.

Nell. J. A. & B. Perkins. 2005. Evaluation of progeny of fourth generation Sydney rock oyster Saccostrea glomerata (Gould 1850) breeding lines. Aquacult. Res. 36:753-757.

Nell. J. A. & B. Perkins. 2006. Evaluation of the progeny of third-generation Sydney rock oyster Saccostrea glomerata (Gould 1850) breeding lines for resistance to QX disease Marteilia sydneyi and winter mortality Bonamia roughleyi. Aquacult. Res. 37:693-700.

NSW Department of Primary Industries. 2004. Aquaculture Production Report 2003/2004. NSW Department of Primary Industries, Port Stephens Fisheries Centre. 20 pp.

O. I. E. 2003. Manual of diagnostic tests for aquatic animals 2003. Paris, France: The World Organisation of Animal Health (Office International des Epizooties).

Richardson, L. B., D. T. Burton & A. M. Stavola. 1982. A comparison of ozone and chlorine toxicity to three life stages of the American oyster Crassostrea virginica. Mar. Environ. Res. 6:99-113.

Roosenburg. W. H., J. C. Rhoderick, R. M. Block, V. S. Kennedy, S. R. Gullans, S. M. Vreenegoor, A. Rosenkranz & C. Collette. 1980. Effects of chlorine-produced oxidants on survival of larvae of the oyster Crassostrea virginica. Mar. Ecol. Prog. Ser. 3:93-96.

Stewart, M. E., W. J. Blogoslawski, R. Y. Hsu & G. R. Helz. 1979. Byproducts of oxidative biocides: toxicity to oyster larvae. Mar. Pollut. Bull. 10:166-169.

Suphamat, K. 2005. Research into the levels of toxicity of Virkon[R] S which affect giant tiger prawns at different stages, http://www. antecint.co.uk/main/prawntox.htm. Accessed December 2005.

White, I. 2002. Safeguarding environmental conditions for oyster cultivation in New South Wales. Report (Number 010801) for the NSW Healthy Rivers Commission. Sydney. 84 pp.

Wilson, S. P. & R. V. Hyne. 1997. Toxicity of acid-sulfate soil leachate and aluminium to embryos of the Sydney rock oyster. Ecotoxicol. Environ. Saf. 37:30-36.

Wisely, B. & R. A. P. Blick. 1967. Mortality of marine invertebrate larvae in mercury, copper, and zinc solutions. Aust. J. Mar. Freshwater Res. 18:63-72.

MICHAEL C. DOVE * AND WAYNE A. O'CONNOR

NSW Department of Primary Industries, Port Stephens Fisheries Centre, Taylors Beach, NSW 2316, Australia

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Details of hatchery substances and the procedure tested in preliminary
range finding tests on S. glomerara embryos.

Substance/ Procedure       Applications

Sodium hypochlorite       Sterilization of tanks,
  (chlorine)                plumbing, equipment,
                            clothes and seawater
Iodine (iodophors)        Sterilization of external surfaces
                            of bivalve broodstock,
                            footwear, equipment, hands
                            and laboratory surfaces
Potassium permanganate    Sterilization of equipment
Virkon S & Virkon S for   Sterilization and cleaning
  Aquaculture               of tanks
Antec Cleaning            Sterilization and cleaning
  and Sanitizing Agent      of tanks
  (DSC-1,000)
Pyroneg                   Cleaning of equipment
Sodium thiosulfate        Neutralization of chlorine
                            and iodine
Rainwater                 Cleaning, rinsing and
                            dilution of seawater for
                            salinity reduction
Bore water                Cleaning, rinsing
                            and dilution of seawater
                            for salinity reduction
Algal supernatant         Food for larvae
Egg screening             Egg screening to rinse,
                            separate or remove
                            feces and debris from
                            spawning

                                        Suggested
Substance/ Procedure                  Concentration

Sodium hypochlorite       ([dagger]) 30 mg available chlorine
  (chlorine)                [L.sup.-1] for sterilizing clean surfaces
                            and in water.
                          ([dagger]) 50 mg available chlorine
                            [L.sup.-1] for pipelines, tanks,
                            equipment, effluent water, clothing, and
                            footbaths (exposure time >30 min)
Iodine (iodophors)        ([dagger]) 200-250 mg iodine [L.sup.-1] (for
                            10 min) for pipelines, tanks,
                            equipment, clothing
Potassium permanganate    ([double dagger]) Approximately 25 g
                            [L.sup.-1]
Virkon S & Virkon S for   ([section]) 5 g [L.up.-1] for cleaning and
  Aquaculture               disinfecting nonporous surfaces
Antec Cleaning            ([section]) 1:1,000 dilution for
  and Sanitizing Agent      cleaning purposes. 1:100-1:800
  (DSC-1,000)               for biocidal treatments
Pyroneg                   ([paragraph]) 2-3 g [L.sup.-1] in warm water
Sodium thiosulfate        ([dagger]) 2.85 times the amount of residual
                            chlorine (grams), and 0.78 times
                            the amount of iodine (grams) used
Rainwater                 ([parallel]) Up to 90% rainwater in seawater
                            for larval experiments
Bore water                ([parallel]) Up to 90% bore water in
                            seawater for larval experiments
Algal supernatant         ([parallel]) 0.1%-0.2% of algal
                            mixture added to larval
                            tanks in the first 48 h of culture
Egg screening             ([parallel]) 1-3 passes
                            of eggs through a 20 Itm to 65 gm
                            mesh screen

                              Type of       Risk of
Substance/ Procedure         Hatchery       Impact *

Sodium hypochlorite       Bivalve, fish &     High
  (chlorine)                crustacean
Iodine (iodophors)        Bivalve, fish &     Low
                            crustacean
Potassium permanganate    Bivalve             Low
Virkon S & Virkon S for   Bivalve, fish &     High
  Aquaculture               crustacean
Antec Cleaning            Bivalve, fish &     Low
  and Sanitizing Agent      crustacean
  (DSC-1,000)
Pyroneg                   Bivalve, fish &     Low
                            crustacean
Sodium thiosulfate        Bivalve, fish &     Low
                            crustacean
Rainwater                 PSFC bivalve        High
                            hatchery
Bore water                PSFC bivalve        High
                            hatchery
Algal supernatant         PSFC bivalve        High
                            hatchery
Egg screening             PSFC bivalve        High
                            hatchery

* To larvae under present practices used at the PSFC bivalve hatchery.

([dagger]) OIE Manual (2003).

([double dagger]) Sigma Pharmaceuticals Pty Limited, Clayton,
Victoria, Austrailia.

([section]) Antec International Limited, Suffolk, UK.

([paragraph]) JohnsonDiversey Australia Pty Limited, Smithfield, NSW,
Australia.

([parallel]) Concentration currently used at the PSFC bivalve
hatchery.

TABLE 2.
Preliminary range finding test exposure levels and proportion of
abnormal development. The mean ([+ or -] 95% confidence interval,
n = 3) proportion abnormal development for the control
treatment was 1.8 [+ or -] 1.5%.

                                                           Abnormal
                                                          Development
Substance/Procedure          Units       Concentrations       (%)

Sodium hypochlorite      mg [L.sup.-1]     0.002               15
  (chlorine)
                                           0.21                11
                                           2.08                92
Iodine (lodophors)       mg [L.sup.-1]     0.0003               9
                                           0.03                12
                                           0.30                13
Potassium permanganate   mg [L.sup.-1]     0.0002              14
                                           0.02                22
                                           0.20                63
Antec Virkon S           mg [L.sup.-1]     0.50                69
                                          50                   97
                                         500                  100
Antec Cleaning                --           1:10000000          39
  and Sanitizing Agent                     1:10000            100
  (DSC-1,000)
Pyroneg                  mg [L.sup.-1]     0.15                32
                                          15.3                100
Sodium thiosulfate       mg [L.sup.-1]     0.0006              28
                                           0.06                26
                                           0.63               100
De-ionized water               %           0.01                 8
                                           1                    4
                                          10                    7
Rainwater                      %           0.01                 9
                                           1                   75
                                          10                  100
Bore water                     %           0.01                11
                                           1                   16
                                          10                   98
Algal supernatant              %           0.01                10
                                           1                    9
                                          10                   15
                                          50                   66
Egg screening            No. of            1                    8
                           repetitions     4                    8
                                           8                   17

TABLE 3.
Definitive toxicity test results for S. glomerata embryos exposed
to substances commonly used for larval rearing at the PSFC bivalve
hatchery.

Toxicant                Concentrations           Date         Test

Rainwater (%)        0, 0.01, 0.01, 1, 10     17 Aug 2004       1
                                              24 Aug 2004       2
                                              24 Aug 2004       3
Bore water (%)          0, 0.01, 1, 10        17 Aug 2004       1
                                              24 Aug 2004       2
                                              24 Aug 2004       3
Algal                0, 0.1, 1, 10, 20, 40    17 Aug 2004       1
  supernatant (%)                             24 Aug 2004       2
                                              24 Aug 2004       3
Chlorine             0, 0.002, 0.02, 0.21,    17 Aug 2004       1
  (mg [L.sup.-1])   0.42, 0.83, 1.66, 20.08   24 Aug 2004       2
                                              24 Aug 2004       3
Virkon S              0, 0.05, 0.5, 5, 50     17 Aug 2004       1
  (mg [L.sup.-1])                             24 Aug 2004       2
                                              24 Aug 2004       3
                                              1 May 2006        4
                                              1 May 2006        5
Virkon[R] S for       0, 0.05, 0.5, 5, 50     1 May 2006        1
  Aquacluture                                 1 May 2006        2
  (mg [L.sup.-1])

Toxicant            [Ec.sub.50] (95% CL)      LOEC          NOEC

Rainwater (%)        0.67 (0.53-0.86)          1             0.10
                     2.29 (1.88-2.77)          1             0.10
                     1.55 (1.24-1.94)          1             0.10
Bore water (%)       3.70 (3.29-4.15)         10             1
                     2.30 (1.93-2.75)         10             1
                      2.9 (2.44-3.45)         10             1
Algal               17.80 (16.64-19.03)       20            10
  supernatant (%)   17.43 (14.60-20.80)       20            10
                     6.51 (5.26-8.06)         10             1
Chlorine             0.76 (0.69-0.84)          0.83          0.42
  (mg [L.sup.-1])    1.11 (1.04-1.18)          1.66          0.83
                     1.18 (1.09-1.28)          1.66          0.83
Virkon S             0.47 (0.35-0.63)          0.5           0.05
  (mg [L.sup.-1])    0.58 (0.43-0.79)          0.5           0.05
                     0.65 (0.42-1.02)          0.05          0
                     0.91 (0.74-1.13)          0.5           0.05
                     1.01 (0.80-1.28)          0.05          0
Virkon[R] S for      1.12 (0.92-1.37)          0.5           0.05
  Aquacluture        0.99 (0.80-1.21)          0.5           0.05
  (mg [L.sup.-1])

TABLE 4.
Chemical analysis * results of seawater (diluent), de-ionized water,
bore water, and rainwater samples collected at the PSFC bivalve
hatchery.

       Detection limits   Seawater (diluent)   De-ionized water
Ion    (mg [L.sup.-1])     (mg [L.sup.-1])     (mg [L.sup.-1])

Al          0.070                 bd                  bd
As          0.006                 bd                  0.02
B           0.006                 bd                  bd
Ba          0.001                 0.01                0.01
Ca          0.147               413.62                0.20
Cd          0.096                 bd                  bd
Co          0.082                 bd                  bd
Cr          0.035                 bd                  bd
Cu          0.001                 0.07                0.03
Fe          0.001                 0.01                 bd
K           0.024               410.21                0.32
Li          0.006                 0.17                 bd
Mg          0.004              1311.30                0.03
Mn          0.016                 bd                  bd
Na          0.000             11254.26                0.44
Ni          0.002                 bd                  bd
P           0.009                 bd                  bd
Pb          0.241                 bd                  bd
Sb          0.261                 bd                  bd
Si          0.048                 bd                  bd
S           0.050              1096.18                0.07
Sr          0.049                 7.59                bd
Ti          1.984                 bd                  bd
Zn          0.024                 0.05                bd

          Bore water          Rainwater
Ion    (mg [L.sup.-1])     (mg [L.sup.-1])

Al            bd                  bd
As            0.01                0.02
B             0.05                bd
Ba            0.02                0.02
Ca            2.93                0.83
Cd            bd                  bd
Co            bd                  bd
Cr            bd                  bd
Cu            0.34                0.25
Fe            bd                  bd
K             4.50                0.94
Li            bd                  bd
Mg            4.94                1.38
Mn            bd                  bd
Na          135.49               11.79
Ni            bd                  bd
P             bd                  bd
Pb            bd                  bd
Sb            bd                  bd
Si            2.08                bd
S            25.07                1.45
Sr            bd                  bd
Ti            bd                  bd
Zn            0.27                2.59

* Samples filtered through 0.22 [micro]m cellulose nitrate filter
paper and analyzed using Inductively Coupled Plasma Atomic Excitation
Spectroscopy (model Perkin Elmer Optima 3000 DV).

bd = below detection unit.